Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myelogenous leukemia.

Constitutive activation of the MEK/ERK pathway is frequently observed in primary acute myelogenous leukemia (AML) samples, but not in normal CD34+ progenitors. ERK promotes cell growth and viability of leukemic cells, and its constitutive activation is an independent prognostic factor for survival in AML patients. We observed that the specific MEK inhibitor CI-1040 impaired cell growth and survival, abrogating clonogenicity of leukemic cells. No data is available on the effects of MEK inhibitors on primary relapsed/refractory AML cells. We applied a flow cytometry technique to quantitate the expression of phosphorylated ERK (p-ERK) in AML samples and normal CD34+ progenitor cells, and evaluated the in vitro effects of CI-1040.We analyzed p-ERK levels in bone marrow or peripheral blood samples from 42 primary AML patients (16 samples collected from AML at diagnosis and 26 from relapsed/refractory AML). Highly purified normal bone marrow and G-CSF mobilized CD34+ progenitors were collected and used as controls. ERK activation was analyzed using a monoclonal antibody specific for p-ERK and Kolmogorov- Smirnov statistics. Results were expressed as D values (D), which allows the objective quantitation of differences in fluorescence intensity. Samples with D > 0.1 were considered positive. In addition, we evaluated changes of p-ERK induced by short-term cell culture (up to 24h) in FCS (10%)-containing medium in the presence or absence of MEK inhibitor (CI-1040, 3uM). Cell viability and phosphatidylserine externalization were determined. Normal bone marrow CD34+ cells at most weakly expressed p-ERK (D=0.07±0.035), while G-CSF mobilized CD34+ cells exhibited markedly levels of p-ERK (D=0.56±0.038). Likewise, marked p-ERK levels were found in 83.3% (35/42) of the AML samples, with a mean D value equal to 0.32±0.04.No differences between samples at diagnosis and relapsed/refractory samples with respect to p-ERK levels were observed: D values were 0.36±0.06 vs. 0.30±0.05 (p=n. s. ). Next, we examined effects of the specific MEK inhibitor CI-1040.In AML cell lines with constitutive ERK phosphorylation, CI- 1040 completely inhibited ERK activation. In primary AML, a statistically significant decrease in p-ERK levels were detected after a 24 hour incubation with CI-1040 D=0.46±0.04 to D=0.31±0.04 (p=0.00027). Newly diagnosis and relapsed/refractory samples were equally sensitive to the inhibitory effects of CI- 1040. CI-1040 did not induce apoptosis compared to vehicle in all but one samples examined. In conclusion, the increase in p-ERK levels that were detected in mobilized normal circulating CD34+ cells was likely due to activation by G-CSF. In contrast, high levels of p-ERK observed in the majority of primary AML samples suggest a disregulation of the MEK/ERK pathway, which is apparently independent of disease stage. CI-1040 inhibited ERK activation in vitro without inducing apoptosis. Consequently, MEK inhibitors may be more effective in combination with cytotoxic agents for the therapeutic modulation of AML. Our previous data have suggested striking synergism with apoptosis modulators